Application of maize ZmRAFS gene to improve crop heat stress tolerance

ABSTRACT

An application of maize raffinose synthase (ZrnRAFS) gene in maize to improve heat stress tolerance of crops is provided. The nucleotide sequence of the ZrnRAFS gene is shown in SEQ ID NO: 1, and the protein sequence encoded by the ZrnRAFS gene is shown in SEQ ID NO: 2. The ZrnRAFS gene is overexpressed in plants, raffinose is synthesized by using sucrose and galactinol, which increases the content of raffinose in maize leaves and improves the heat stress tolerance of maize.

TECHNICAL FIELD

The disclosure relates to the field of plant biotechnology, and moreparticularly to a key enzyme gene ZmRAFS (GRMZM2G150906) encodingraffinose synthase that are responsible for synthesis of raffinose andan application/use thereof.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the XML file containingthe sequence listing is 23060TBYX-USP1-SL.xml. The XML file is 7,577bytes; is created on Jul. 24, 2023; and is being submittedelectronically via EFS-Web.

BACKGROUND

A previous research achievement of the inventor discloses thatoverexpression of maize raffinose synthase (ZmRAFS) gene from Maize(also referred to as Zea mays) in Arabidopsis thaliana improves plantdrought tolerance by hydrolyzing galactinol to produce inositol (Li, T.,et al., Raffinose synthase enhances drought tolerance through raffinosesynthesis or galactinol hydrolysis in maize and Arabidopsis plants.Journal of Biological Chemistry. 2020. 295:8064-8077.). Droughttolerance is the ability of a plant to survive or grow in an environmentadapted to growth temperature (e.g., around 28° C.), under short-term orlong-term water shortage.

SUMMARY

The inventor finds that overexpression of maize raffinose synthase(ZmRAFS) gene (GRMZM2G150906) in maize plants increases the content ofraffinose in leaves and enhances the heat stress tolerance of theplants.

Based on this, the disclosure provides an application of ZmRAFS gene inmaize to improve heat stress tolerance of crops, and the sequence of theZmRAFS gene is shown in SEQ ID NO: 1. The protein sequence encoded bythe ZmRAFS gene is shown in SEQ ID NO: 2.

In some embodiments, the enzyme encoded by the ZmRAFS gene usesgalactinol and sucrose as substrates to synthesize raffinose in theleaves, and the heat stress tolerance of the plants is improved byregulating the content of raffinose in the leaves. In an embodiment, thecrop is maize.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a schematic diagram of a maize overexpression vector;where pUbi represents a Maize Ubiquitin promoter, ZmRAFS represents amaize raffinose synthase gene, NOS represents a terminator, p35Srepresents a Cauliflower mosaic virus (CaMV) 35S promoter, Barrepresents an herbicide-resistance gene, Tvsp represents anotherterminator, and HindIII and EcoRV represent restrictive enzyme sites.

FIGS. 2A-2D illustrate that the content of raffinose in leaves of maizeplants overexpressing ZmRAFS is significantly increased. Specifically,FIG. 2A illustrates identification of overexpressed maize plants atgenomic level; where Marker represents DNA molecular weight, Controlrepresents polymerase chain reaction (PCR) water control (also referredto as blank control), Zong31 represents maize Zong31 inbred line genome,Plasmid represents Plasmid DNA, used as a positive control, PTF101.1ZmRAFS (1 . . . 9) represent 9 independent transformation eventsoverexpressing ZmRAFS. FIG. 2B illustrates identification of ZmRAFS mRNAlevel in leaves of overexpressed maize plants. FIG. 2C illustratesidentification of ZmRAFS protein level in the leaves of theoverexpressed maize plants; where Zong31 represents maize Zong31 inbredline, ZmRAFS #1, #2, #3 represent three plants overexpressing the ZmRAFSgene (background of Zong31), ZmGAPDH representsglyceraldehyde-3-phosphate dehydrogenase gene, as an internal reference.FIG. 2D illustrates determination of the content of raffinose in theleaves of the overexpressed maize plants; where Zong31 represents maizeZong31 inbred line, ZmRAFS #1, #2, #3 represent three plants overexpressing the ZmRAFS gene (background of Zong31), data representsmean±standard error, n=3, and * represents significant difference(Student's t-test, * P<0.05, ** P<0.01).

FIGS. 3A-3C illustrate that overexpression of ZmRAFS gene significantlyimproves heat stress tolerance of the maize plants. Specifically, FIG.3A illustrates growth phenotypes of maize plants overexpres sing theZmRAFS gene and control plants under normal, heat shock treatment, andrecovery, with a scale of 5 centimeters (cm). FIG. 3B illustrates thatthe survival rate of the maize plants overexpres sing the ZmRAFS geneafter heat stress treatment is significantly higher than that of thecontrol plants. FIG. 3C illustrates that the conductivity of the maizeplants overexpres sing the ZmRAFS gene is significantly lower than thatof the control plants after heat stress treatment; where Zong31represents maize Zong31 inbred line; ZmRAFS (#1, #2, #3) representZmRAFS transgenic plants, data represents mean±standard error, eachblack dot represents one biological repeat (8 seedlings), and each planthas eight biological repeats, and different small letters representsignificant differences (Duncan test, P<0.05).

DETAILED DESCRIPTION OF EMBODIMENTS

Unless otherwise specified, scientific and technological terms andmethods herein are understood by those skilled in the related art or areimplemented by methods well-known to those skilled in the related art.The heat stress tolerance is the ability of plants to survive or grow ata high temperature (above 30° C.). The technical solution of thedisclosure will be further described below with reference to theaccompanying drawings and specific embodiments. Unless otherwisespecified, the materials or reagents used in the following embodimentsare all commercially available products.

Embodiment

In this embodiment, the coding region of maize ZrnRAFS gene is cloned bypolymerase chain reaction (PCR), a plant expression vector of the ZmRAFSgene is constructed, maize is transformed, and a maize transgenic plantis obtained. The method is specifically described as follows.

-   -   (1) RNA is extracted from leaves of B73 maize seedlings at the        three-leaf stage and reverse transcribed into complementary DNA        (cDNA).    -   (2) Using cDNA as a template, the coding region of ZmRAFS shown        in SEQ ID NO: 1 is amplified with an upstream primer 5′        -CGCGGATCCATGGCTCCCACCACCAGCCAAGAG-3′ as shown in SEQ ID NO: 3        and a downstream primer        5′-TGCTTAGAGGTAGAGAGAGAGAGAGAGACGACTGAGGGACGACAGAG-3′ as shown        in SEQ ID NO: 4. The amplification procedures are:        pre-denaturation at 95° C. for 5 minutes; 95° C. denaturation        for 30 seconds, annealing at 60° C. for 30 seconds, extension at        72° C. for 1 minute and 20 seconds, 35 cycles; and final        extension at 72° C. for 10 minutes.    -   (3) The amplification product is purified, digested and        recovered, and then ligated to the maize expression vector        pTF101. After that, the vector is transformed into an        Agrobacterium AGL1 strain, as shown in FIG. 1 .    -   (4) The obtained Agrobacterium AGL1 strain is used for genetic        transformation of maize, and the maize transformation receptor        is the Zong31 inbred line (Hui Yang, Guoying Wang and Jingrui        Dai, 2001, Transformation of maize inbred line Zong3 and Zong31,        Journal of Agricultural Biotechnology,        9(04):334-337.doi:10.3969/j.issn.1674-7968.2001.04.008). Nine        transgenic positive plants are obtained by infecting immature        embryos of Zong31 at 10 days after pollination.

In addition, molecular characterization of three of the above ninetransgenic positive plants (#1, #2 and #3) is performed. It is foundthat the expression of mRNA and protein of the ZmRAFS gene in the leavesof the transgenic plants is increased compared with that of the control,and the content of raffinose is significantly higher than that of thecontrol. The method is specifically described as follows.

-   -   (a) The insertion and the mRNA expression of the transgene of        the transgenic positive plants are identified by PCR and RT-PCR.        The PCR amplification procedures are: pre-denaturation at 95° C.        for 5 minutes; denaturation at 95° C. for 30 seconds, annealing        at 60° C. for 30 seconds, extension at 72° C. for 30 seconds, 28        cycles; and final extension at 72° C. for 8 minutes; and the        results are shown in FIGS. 2A and 2B.    -   (b) The protein accumulation of the ZmRAFS of the transgenic        positive plants is identified by Western blotting, the        procedures of Western blotting refer to the method published in        the literature: Gu, L., et al., ZmGOLS2, a target of        transcription factor ZmDREB2A, offers similar protection against        abiotic stress as ZmDREB2A. Plant Mol Biol, 2016.90(1-2):p.        157-70, and the ZmRAFS primary antibody used in the method is        prepared according to the method published in the literature:        Li, T., et al., Regulation of seed vigor by manipulation of        raffinose family oligosaccharides (RFOs) in maize and        Arabidopsis. Molecular Plant. 2017. 10(12): 1540-1555, and the        dilution ratio is 1: 5000. The secondary antibody (goat        anti-rabbit IgG) is purchased from Jiangsu Cowin Biotech Co.,        Ltd, and the dilution ratio is 1: 10000. The results are shown        in FIG. 2C.    -   (c) The content of raffinose in the leaves of the above plants        #1, #2, #3 and a wild-type control plant is identified by        high-performance liquid chromatography (HPLC), the method refers        to the literature (Li, T., et al., Raffinose synthase enhances        drought tolerance through raffinose synthesis or galactinol        hydrolysis in maize and Arabidopsis plants. Journal of        Biological Chemistry. 020. 295:8064-8077); and the results are        shown in FIG. 2D.

SEQ ID NO: 1 is as follows:atggctccca acctcagcaa gaagacgcct gctggcctcc tcggcgacga ggtggccccg   60gtggagggac tcaagccgtc gcggttcacc cttaagggca aggacctggc cgtggacggg  120cacccggtcc tgctggacgt gccggccaac atccgtctca ccccggcgtc gacgctcgtg  180cccgccgcgg acgtccccgc agcgggcggc ggcagcttcc tcggcttcga cgcggcggcg  240gccgagagcc ggcacgtggt gcccgtcgga aagctccgtg acattcggtt catgagcatc  300ttccgtttca aggtgtggtg gacgacgcac tgggtggggg acagcggcag ggacgtggag  360aacgagacgc agatgatggt gctcgaccgc tccgccggcg agcccggcgg cggcggccga  420ccctacgtgc tgctgctccc catcatcgag ggctcgttcc gggcctgcct cgaggccggg  480aaggtggaag actacgtgga cctgtgcgtg gagagcgggt cgtcggcggt gcgcggcgcc  540gcgttccgga gctcgctgta cctgcacgcg ggcgacgacc cgttcgagct cgtcgcggac  600gccgtcaggg tggtccgtgc gcacctgggc acgttccgga ccatggagga gaagacgccg  660ccgccgatcg tggacaagtt cgggtggtgc acgtgggacg ccttctacct caaggtgcac  720ccggagggcg tgtgggaggg cgtgcgccgc ctggcggagg gcggctgccc gccggggctg  780gtgctcatcg acgacggctg gcagtccatc tgccacgacg aggacgaccc gaacagcggc  840gaggagggca tgaaccgcac ctccgccggc gagcagatgc cctgccgcct catcaagttc  900caggagaacc acaagttcag ggagtacaag cagggcggga tgggcgcgtt cgtgcgggag  960atgaaggcgg cgttccccac cgtggagcag gtgtacgtgt ggcacgcgct gtgcgggtac 1020tggggcggcc tccgccccgg cgcgcccggc ctgccgcccg ccaaggtggt ggcgcccaag 1080ctctcccccg gcctgcagcg caccatggag gacctcgccg tcgacaagat cgtcaacaac 1140ggtgtcggcc tcgtcgaccc caagcgcgcg cacgagctct acgatggttt gcactcccac 1200ctccaggcct ccggcatcga cggcgtcaag gtcgacgtca ttcacttgct ggagatgctg 1260tgcgaggagt acggcggccg tgtcgagctg gccaaggcct acttcgccgg gctgacggcg 1320tcggtgcggc ggcacttcgg cggcaacggc gtgatcgcga gcatggagca ctgcaacgac 1380ttcatgctgc tgggcacgga ggcggtggcg ctgggccgcg tgggcgacga cttctggtgc 1440acggacccct ccggcgaccc caacggcacc ttctggctgc aggggtgcca catggtgcac 1500tgcgcctaca actcgctgtg gatgggcaac ttcatccacc cggactggga catgttccag 1560tccacgcacc cctgcgccgc cttccacgcc gcgtcccgcg ccatctccgg cgggcccatc 1620tacgtcagcg actcggtggg gcagcacgac ttcgcgctgc tccgccgcct ggcgctcccc 1680gacggcaccg tcctccggtg cgagggccac gcgctgccca cgcgcgactg cctcttcgcc 1740gacccgctcc acgacggccg gaccgtgctc aagatctgga acgtgaaccg cttcgccggc 1800gtcgtcggcg ccttcaactg ccagggcggc gggtggagcc ccgaggcgcg gcggaacaag 1860tgcttctcgg agttctccgt gcccctggcc gcgcgcgcct cgccgtccga cgtcgaatgg 1920aagagcggca aagcgggacc aggcgtcagc gtcaagggcg tctcccagtt cgccgtgtac 1980gcggtcgagg ccaggacgct gcagctgctg cgccccgacg agggcgtcga cctcacgctg 2040cagcccttca cctacgagct cttcgtcgtc gcccccgtgc gcgtcatctc gcacgagcgg 2100gccatcaagt tcgcgcccat cggactcgcc aacatgctca acaccgccgg cgccgtgcag 2160gcgttcgagg ccaagaaaga tgctagcggc gtcacggcag aggtgttcgt gaagggcgca 2220ggggagctgg tggcgtactc gtcggcgacg cccaggctct gcaaggtgaa cggcgacgag 2280gccgagttca cgtacaagga cggcgtggtc accgtcgacg tgccgtggtc ggggtcgtcg 2340tcgaagctgt gtcgcgtcca gtacgtctac tga.                             2373SEQ ID NO: 2 is as follows: MAPNLSKKTPAGLLGDEVAPVEGLKPSRFTLKGKDLAVDGHPVLLDVPANIRLTPASTLVPAADVPAAGGGSFLGFDAAAAESRHVVPVGKLRDIRFMSIFRFKVWWTTHWVGDSGRDVENETQMMVLDRSAGEPGGGGRPYVLLLPIIEGSFRACLEAGKVEDYVDLCVESGSSAVRGAAFRSSLYLHAGDDPFELVADAVRVVRAHLGTFRTMEEKTPPPIVDKFGWCTWDAFYLKVHPEGVWEGVRRLAEGGCPPGLVLIDDGWQSICHDEDDPNSGEEGMNRTSAGEQMPCRLIKFQENHKFREYKQGGMGAFVREMKAAFPTVEQVYVWHALCGYWGGLRPGAPGLPPAKVVAPKLSPGLQRTMEDLAVDKIVNNGVGLVDPKRAHELYDGLHSHLQASGIDGVKVDVIHLLEMLCEEYGGRVELAKAYFAGLTASVRRHFGGNGVIASMEHCNDFMLLGTEAVALGRVGDDFWCTDPSGDPNGTFWLQGCHMVHCAYNSLWMGNFIHPDWDMFQSTHPCAAFHAASRAISGGPIYVSDSVGQHDFALLRRLALPDGTVLRCEGHALPTRDCLFADPLHDGRTVLKIWNVNRFAGVVGAFNCQGGGWSPEARRNKCFSEFSVPLAARASPSDVEWKSGKAGPGVSVKGVSQFAVYAVEARTLQLLRPDEGVDLTLQPFTYELFVVAPVRVISHERAIKFAPIGLANMLNTAGAVQAFEAKKDASGVTAEVFVKGAGELVAYSSATPRLCKVNGDEA EFTYKDGVVTVDVPWSGSSSKLCRVQYVY.

The specific method for identifying heat stress tolerance of thetransgenic positive plants obtained from the embodiment is as follows.

-   -   (i) Maize seedling cultivation method is as follows. The ZmRAFS        transgenic plants (#1, #2, #3) and the wild-type control plant        (Zong31) are planted in an incubator. Seeds of each experimental        group are soaked and germinated in darkness at 28° C. for 72        hours, the maize with the same growth vigor (root length of        about 2-3 cm) is selected and transplanted into the pot, with 8        plants in each pot. When transplanting, the nutrient soil and        water are mixed in a weight ratio of 1:1, the weight of each pot        is controlled to 170 grams (g). After marking, the maize is        placed in the incubator and grown in darkness at 28° C. for 16        hours under light (10,000 LUX)/8 hours.    -   (ii) Seedling heat shock treatment and survival rate statistics        are as follows. When the seedlings reach the three-leaf stage,        they are subjected to heat shock treatment at 42° C. When the        leaves of the control group are irreversibly rolled (the rolled        leaves cannot be restored the next morning), they are        photographed and recorded, and the phenotype is observed and        photographed after recovery culture at 28° C., as shown in FIG.        3A. The survival rate is calculated and the conductivity is        measured. Survival rate=number of surviving plants/total number        of plants x 100%; and the results are shown in FIG. 3B.    -   (iii) Conductivity measurement is as follows. The leaves of        ZmRAFS transgenic maize plants and the control plant after heat        shock recovery are taken for conductivity measurement. The        leaves of the same leaf position are soaked in 15 mL of        deionized water, vacuumed for 30 minutes, treated at 25° C. and        120 revolutions per minute (rpm) for 1 hour, and the        conductivity is recorded as C₁. After the leaves are bathed in        boiling water for 30 minutes and then cooled to room        temperature, the conductivity (also referred to as ion leakage)        is measured and recorded as C₂; C₁/C₂×100% is the relative        conductivity of the leaves. The conductivity meter model is        Thunder Magnet DDS-307; and the results are shown in FIG. 3C.

The above is only the illustrated embodiment of the disclosure, and thescope of protection of the disclosure is not limited to this. Any personskilled in the related art can obviously obtain simple changes orequivalent substitutions of the technical solution within the scope ofthe technology disclosed herein, which all belong to the scope ofprotection of the disclosure.

What is claimed is:
 1. An application method of maize raffinose synthase(ZrnRAFS) gene in maize, comprising: using the ZrnRAFS gene to improveheat stress tolerance of crops, wherein a sequence of a ZrnRAFS gene isshown in SEQ ID NO:
 1. 2. The application method as claimed in claim 1,wherein a protein sequence encoded by the ZrnRAFS gene is shown in SEQID NO:
 2. 3. The application method as claimed in claim 1, comprising:regulating content of raffinose in leaves of the corps throughmanipulation of the ZmRAFS gene to thereby improve the heat stresstolerance of the crops.
 4. The application method as claimed in claim 1,wherein the crop is maize.
 5. The application method as claimed in claim1, comprising: extracting ribonucleic acid (RNA) from leaves of maizeseedlings at a three-leaf stage and reverse transcribing into copydeoxyribonucleic acid (cDNA); amplifying a coding region of the ZmRAFSgene by using the cDNA as a template with an upstream primer shown inSEQ ID NO: 3 and a downstream primer shown in SEQ ID NO: 4 to obtain anamplification product; ligating the amplification product to a maizeexpression vector and transforming the ligated maize expression vectorinto an Agrobacterium strain; and performing genetic transformation ofmaize on the transformed Agrobacterium strain to a maize transformationreceptor, so as to obtain a target plant with the heat stress tolerance.6. The application method as claimed in claim 5, wherein procedures ofthe amplifying comprise pre-denaturation at 95° C. for 5 minutes; 95° C.denaturation for 30 seconds, annealing at 60° C. for 30 seconds,extension at 72° C. for 1 minute and 20 seconds, 35 cycles; and finalextension at 72° C. for 10 minutes.